Gemcitabine (dFdCyd) is an analog of cytosine arabinoside with clinical activity against a variety of solid tumors. Based on our preclinical studies demonstrating that dFdCyd is a potent radiation (RT) sensitizer, we designed a novel clinical trial combining dFdCyd with RT for the treatment of patients with advanced head and neck cancers. This trial has produced pathologic complete responses in the first 10/11 evaluable patients, although there has been substantial toxicity. The long term goal of this proposal is to understand the mechanism of dFdCyd-mediated radiosensitization and to use this information, in combination with data from animal models and biopsies from patients' tumors, to increase the efficacy of dFdCyd as a RT sensitizer. This goal will be addressed through 4 specific aims: Specific Aim 1 is to determine the roles of nucleotide pool depletion and cell cycle redistribution in radiosensitization. We hypothesize that inhibition of ribonucleotide reductase leading to dATP pool depletion and redistribution into S phase are the critical factors in producing dFdCyd-mediated radiosensitization. Specific Aim 2 is to elucidate the mechanism by which dFdCyd increases RT-induced apoptosis. Our preliminary results show that apoptosis plays a key role in dFdCyd-mediated radiosensitization. We hypothesize that pre-treatment with dFdCyd with the accompanying effects of pool depletion and S phase redistribution increase RT-induced apoptosis. In Specific Aim 3, we propose to use athymic nude mice bearing xenografts to determine if pharmacological and cell cycle redistribution parameters can predict tumor growth delay resulting from combined treatment with dFdCyd and RT. We hypothesize that the greatest antitumor effects will be produced under conditions which cause dATP pool depletion and cell cycle redistribution into S phase after treatment with dFdCyd. We also ask if twice weekly infusions of dFdCyd combined with RT produce a better therapeutic index than the once weekly schedule currently used. We hypothesize that RT combined with twice weekly dFdCyd is superior. Specific Aim 4 will assess the effect of dFdCyd in patient biopsies. Biopsies obtained from the tumors of our patients treated with dFdCyd demonstrate that phosphorylated metabolites can be detected. We propose to expand our biopsy studies. We hypothesize that radiosensitization in patient tumors is associated with the accumulation of phosphorylated metabolites of dFdCyd and dATP pool depletion. We feel our preliminary data, research team, and record for translating preclinical findings to the clinic suggest that this proposal has a high likelihood of generating data which will improve the outcome of treatment of patients with unresectable head and neck cancer.